The integrated stress response

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1 Review The integrated stress response Karolina Pakos-Zebrucka 1,2,, Izabela Koryga 1,2,, Katarzyna Mnich 1,2, Mila Ljujic 1,2, Afshin Samali 1,2 & Adrienne M Gorman 1,2,* Abstract In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eif2a) by one of four members of the eif2a kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress. Keywords activating transcription factor 4; eif2a kinase; eukaryotic translation initiation factor 2 alpha; integrated stress response DOI /embr Received 12 February 2016 Revised 3 August 2016 Accepted 23 August 2016 Published online 14 September 2016 EMBO Reports (2016) 17: See the Glossary for abbreviations used in this article. Introduction The integrated stress response (ISR) is an elaborate signaling pathway present in eukaryotic cells, which is activated in response to a range of physiological changes and different pathological conditions [1 5]. Such stresses commonly include cell extrinsic factors such as hypoxia, amino acid deprivation, glucose deprivation, and viral infection [2,4 8]. However, cell intrinsic stresses such as endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins in the ER, can also activate the ISR [9]. Furthermore, in the context of cancer biology, the ISR can be triggered by oncogene activation [10,11]. The common point of convergence for all the stress stimuli that activate ISR is phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eif2a) on serine 51 [1]. In mammalian cells, this is catalyzed by a family of four serine/threonine (S/T) eif2a kinases that are activated by distinct stress stimuli [12]. eif2a phosphorylation causes a reduction in global protein synthesis while allowing the translation of selected genes including activating transcription factor 4 (ATF4), aiding cell survival and recovery [13]. However, if the cellular stress is severe, either in intensity or in duration, it will overwhelm the capacity of the adaptive response to resolve it and additional components become activated to execute cell death. Dephosphorylation of eif2a signals termination of the ISR and return to normal protein synthesis (Fig 1) [14 16]. It is likely that the duration and level of eif2a phosphorylation, as well as ATF4 regulation and its interactions with other proteins, determine the ultimate ISR outcome resulting from different environmental and physiological stresses [16 18]. Here, we outline current knowledge of the molecular mechanisms of the ISR, highlighting the importance of eif2a phosphorylation/dephosphorylation and how signaling events downstream of ATF4 ultimately regulate the cell responses to the particular stress insult. We also discuss the ISR pathway in the context of cell survival and cell death in mammalian cells, and describe pharmacological modifications of the pathway that may have therapeutic value. Activation of the integrated stress response The eif2a kinases act as early responders to disturbances in cellular homeostasis. There are four members of the family: PKR-like ER kinase (PERK) [19], double-stranded RNA-dependent protein kinase (PKR), heme-regulated eif2a kinase (HRI), and general control nonderepressible 2 (GCN2) (Fig 1) [5,12]. All four eif2a kinases share extensive homology in their kinase catalytic domains, but possess distinct regulatory domains [9,20 24]. Each eif2a kinase dimerizes and autophosphorylates for full activation [25]. However, each kinase responds to distinct environmental and physiological stresses (Fig 1), which reflects their unique regulatory mechanisms [12]. For example, PERK, found in metazoans, is located in the endoplasmic reticulum (ER) membrane where its luminal domain is normally bound by 78-kDa glucose-regulated protein (GRP78). ER stress (e.g. due to the accumulation of unfolded proteins in the ER, or perturbations in cellular energy, calcium homeostasis, or redox status) causes the activation of PERK. Two models for the activation of PERK by ER stress have been put forward [26,27]. The classical 1 Apoptosis Research Centre, National University of Ireland Galway, Galway, Ireland 2 School of Natural Sciences, National University of Ireland Galway, Galway, Ireland *Corresponding author. Tel: ; adrienne.gorman@nuigalway.ie These authors contributed equally to this work 1374 EMBO reports Vol 17 No ª 2016 The Authors

2 Karolina Pakos-Zebrucka et al The integrated stress response EMBO reports Glossary b-trcp F-box protein b-transducin repeat-containing protein 5 0 UTR 5 0 untranslated region AARE Amino acid response element Akt Protein kinase B ALS Amyotrophic lateral sclerosis AP-1 Activator protein 1 APAF-1 Apoptotic peptidase-activating factor 1 ASNS Asparagine synthetase ATF/CREB Activating transcription factor/cyclic AMP response element binding protein ATF Activating transcription factor Atg Autophagy-related protein BBC3 p53 upregulated modulator of apoptosis BCL-2 B-cell lymphoma 2 Becn1 Beclin-1 BIM (BCL2L11) BCL-2 interacting mediator of cell death BNIP3L BCL2/adenovirus E1B 19-kDa protein-interacting protein 3-like bzip Basic leucine zipper domain C/EBPb CCAAT/enhancer-binding protein beta CARE C/EBP-ATF response element CDS Coding sequence C H 1 Constant heavy chain domain 1 CHOP C/EBP homologous protein CK1 Casein kinase 1 CK2 Casein kinase 2 CLOCK Circadian locomotor output cycles protein kaput CMT Charcot Marie Tooth disease CReP Constitutive repressor of eif2a phosphorylation DR5 Death receptor 5 eif Eukaryotic translation initiation factor ER Endoplasmic reticulum ERO1a Endoplasmic oxidoreductin-1-like protein FOXO Forkhead box O Gabarap Gamma-aminobutyric acid receptor-associated protein Gabarapl2 Gamma-aminobutyric acid receptor-associated protein-like 2 GADD34 Growth arrest and DNA damage-inducible protein GADD34 GCN2 General control non-depressible protein 2 GRP78 78-kDa glucose-regulated protein HIV Human immunodeficiency virus HRI Heme-regulated eif2a kinase HSP70 and 90 Heat shock proteins 70 and 90 HSV Herpes simplex virus IAPs Inhibitor of apoptosis proteins IBTKa a-isoform of inhibitor of Bruton s tyrosine kinase IRE1 Inositol-requiring enzyme-1 IRES Internal ribosome entry site ISRIB Integrated stress response inhibitor ISR Integrated stress response Map1lc3b (LC3) Microtubule-associated proteins 1 light chain 3 beta MCL-1 Myeloid cell leukemia-1 MEFs Mouse embryonic fibroblasts mtorc1 mtor complex 1 mtor Mammalian target of rapamycin NAIP Neuronal apoptosis inhibitory protein NBR1 Neighbor of BRCA1 gene 1 NF-κB Nuclear factor kappa light chain enhancer of activated B cells NRF2 Nuclear factor erythroid 2-related factor 2 NSRE-1 Nutrient-sensing response elements 1 NSRE-2 Nutrient-sensing response elements 2 NUPR1 Nuclear protein 1 OLA1 Obg-like ATPase 1 p300 Histone acetyltransferase p300 PDX1 Pancreatic and duodenal homeobox 1 PERK PKR-like ER kinase PHD1 Prolyl hydroxylase 1 PHD3 Prolyl hydroxylase 3 PI3K Phosphatidylinositol-3 kinase PIC Pre-initiation complex PKA Protein kinase A PKR Double-stranded RNA-dependent protein kinase PMAIP1 Phorbol-12-myristate-13-acetate-induced protein 1 PP1c Protein phosphatase 1 catalytic subunit PP1 Protein phosphatase 1 PPP1R15A Protein phosphatase 1 regulatory subunit 15A PPP1R15B Protein phosphatase 1 regulatory subunit 15B REDD1 (Ddit4) Regulated in development and DNA damage response 1 Rpl7 60S ribosomal protein L7 RSK2 Ribosomal protein S6 kinase alpha-2 SCF btrcp SCF E3 ubiquitin ligase complex Siah 1/2 E3 ubiquitin protein ligases 1 and 2 Sqstm1 Sequestosome 1 SyK Spleen-associated tyrosine kinase TFE3 Transcription factor E3 TFEB Transcription factor EB TNFRSF10B TNF receptor superfamily member 10b TRAIL TNF-related apoptosis-inducing ligand TRIB3, SKIP3 Tribbles homolog 3 TXNIP Thioredoxin-interacting protein uorf Upstream open reading frame UPR Unfolded protein response VEGF Vascular endothelial growth factor WARS Tryptophanyl-tRNA synthetase XBP1 X-box binding protein 1 XIAP X chromosome-linked inhibitor of apoptosis ZIPK Zipper-interacting protein kinase model proposes that upon the accumulation of misfolded or unfolded proteins in the ER lumen, GRP78 becomes dissociated from PERK, leading to its autophosphorylation and activation [9,22,28,29]. However, recent research suggests an alternative mechanism whereby PERK may be activated directly by binding of unfolded or misfolded proteins to its luminal domain [26,30]. While there is somewhat limited direct evidence for the latter model of PERK activation, it is supported by evidence from another ER sensor of unfolded proteins, inositol-requiring enzyme-1 (IRE1), whose ER luminal domain exhibits strong sequence homology with that of PERK and which in yeast has been shown to be directly activated by unfolded or misfolded proteins [26,30]. PERK activation in response to glucose deprivation has been reported in neuronal cells and in pancreatic b cells [31,32] where it was shown that it is the ATP depletion that leads to the activation of PERK through the inhibition of sarcoplasmic/er Ca 2+ -ATPase pump. It is also interesting to note that in cancer cells, oncogene activation can trigger the ISR through PERK. For example, amplified HRAS induces PERK/eIF2a/ATF4 and cellular senescence in a premalignant model of melanoma [10] and increased activity of proto-oncogene MYC elicits the activation of the PERK/eIF2a/ATF4 resulting in cellular transformation and tumorigenesis [11]. ª 2016 The Authors EMBO reports Vol 17 No

3 EMBO reports The integrated stress response Karolina Pakos-Zebrucka et al Stress signal ISR regulator ISR core ISR effector ISR execution Amino acid deprivation GCN2 GCN2 PP1 CReP Cap-dependent mrna translation INTEGRATED STRESS RESPONSE Viral infection PKR PKR elf2α Dimerization partner ATF4 ATF4 Heme deprivation HRI HRI P elf2α ER stress PERK PERK PP1 GADD34 GADD34 Gene expression Figure 1. Integrated stress response signaling. ER stress, viral infection, and other cellular stress signals activate PERK, PKR, HRI, and GCN2 kinases that converge on phosphorylation of eif2a, the core of ISR. This leads to global attenuation of Cap-dependent translation while concomitantly initiates the preferential translation of ISR-specific mrnas, such as ATF4. ATF4 is the main effector of the ISR. It forms homo- and heterodimers that bind to DNA targets to control the expression of genes involved in cellular adaptation. Termination of the ISR is regulated by the constitutively expressed CReP and stress-inducible phosphatase GADD34 that dephosphorylate eif2a. For more details, see main text and Table 1. Arrows denote activation or induction, while blunt lines indicate inhibition. GCN2 is highly conserved from yeast to humans [33]. Mechanistic insights into GCN2 regulation, largely derived from studies in yeast, indicate that GCN2 binds to deacylated transfer RNAs (trnas) via a histidyl-trna synthetase-related domain to become active in response to amino acid deprivation [34]. GCN2 activation has also been reported in response to a prolonged glucose deprivation in tumor cells, although this is probably an indirect effect resulting from the consumption of amino acids as an alternative energy source in the absence of glucose [7]. GCN2 can also be activated by UV light in mouse embryonic fibroblast (MEF) cells and human keratinocytes [35,36]. The mechanism of GCN2 activation by UV light is unclear and at least two models have been suggested, including UV-induced cross-linking between trna and GCN2, or the rapid consumption of arginine due to UV activation of nitric oxide synthetase [33,35,36]. The mammalian PKR is activated mainly by double-stranded RNA (dsrna) during viral infection [37,38]. Dimerization of PKR via its C-terminal kinase domain results in autophosphorylation at T446 and the subsequent functional activation of the kinase [39 41]. Thus, the presence of dsrna during viral infection results in PKR activation and the subsequent inhibition of viral and host protein synthesis through eif2a phosphorylation [37,42,43]. Interestingly, other stresses such as oxidative and ER stress [44,45], growth factor deprivation, cytokine or bacterial infection [46,47], ribotoxic stress [48], stress granules [49], and heparin [50] have also been shown to stimulate PKR, but in a dsrna-independent manner. Interestingly, PKR can also be stimulated by caspase activity in the early stage of apoptosis, indicating a role for the inhibition of protein synthesis in this cell death program [51]. Unlike the other eif2a kinases that have a broad tissue distribution, HRI is mainly expressed in erythroid cells where it is involved in erythrocyte differentiation during erythropoiesis [52]. HRI couples the translation of globin mrnas with the availability of heme for the production of hemoglobin and thus protects erythroid cells against the accumulation of toxic globin aggregates in iron deficiency [52 55]. Similar to the other eif2a kinases, HRI activation requires dimerization and autophosphorylation of its kinase domain [56,57]. Regulation of HRI kinase activity by heme is mediated by the two heme-binding domains that are located in the N-terminus and the kinase insertion domain [56,57]. Binding of heme to the N-terminus is stable and it co-purifies with HRI, whereas heme binding to the kinase insertion domain is reversible and inhibits HRI kinase activity [54,56]. Heme inhibits HRI kinase activity in vitro and in vivo by promoting disulfide bond formation between HRI monomers, keeping them in an inactive dimer conformation [52,58,59]. However, in the absence of heme, non-covalent interactions between HRI molecules occurs, resulting in an active HRI dimer [52,58,59]. HRI can also be activated by other stresses including arsenite-induced oxidative stress, heat shock, osmotic stress, 26S proteasome inhibition, and nitric oxide [52,60 63]. Interestingly, activation of HRI by these diverse stresses is independent of heme and occurs with the aid of heat shock proteins HSP90 and HSP70; however, the exact mechanism of HRI activation remains to be investigated [63]. The eif2a kinases have overlapping functions and, as such, can act cooperatively to specifically tune cellular responses to a wide variety of stressors, hereafter referred as cellular stress, unless otherwise stated. For example, GCN2 contributes to ER stressinduced eif2a phosphorylation in Perk / MEF cells [64]. Conversely, PERK can compensate for Gcn2 loss in a genetically engineered mouse model of soft tissue sarcoma [65] and in a Gcn2-knockout 5XFAD mouse model of Alzheimer s disease [66], as well as for Hri loss in mouse hepatocytes [58]. Further support for signaling redundancy between PERK and GCN2 comes from experiments on HeLa cells stressed by protein overload where knockdown of either PERK or GCN2 was compensated by a rapid upregulation of the other kinase [67]. Additionally, both PERK and GCN2 become activated during nucleofection [68] and together with PKR can regulate host response to viral infection [8,69,70]. All eif2a kinases become activated in response to oxidative stress [2,55,71,72]. PERK and PKR kinases activate the ISR to effectively manage heat stress and to limit the aggregation and accumulation of the denatured proteins in the ER of human endothelial cells and MEFs, respectively [73,74]. Possibly, in such cases when eif2a kinases act cooperatively, the cellular response is determined not only by ISR activation but also by the activation of other specific substrates of eif2a kinases. Recently, it has been reported that in in vitro models of several types of tumors, upon ER stress, amino acid starvation, and oxidative 1376 EMBO reports Vol 17 No ª 2016 The Authors

4 Karolina Pakos-Zebrucka et al The integrated stress response EMBO reports stress, the ISR can be also modulated by OLA1, a GTPase that inhibits de novo formation of the eif2 ternary complex, providing a secondary mechanism for the inhibition of global mrna translation while permitting ATF4 protein synthesis [75]. Termination of the ISR Dephosphorylation of eif2a is central to ISR signal termination to restore protein synthesis and normal cell functioning [15]. It is mediated by protein phosphatase 1 (PP1) complex that recruits a PP1 catalytic subunit (PP1c) and one of the two regulatory subunits. In mammals, phosphatase activity is regulated by either PPP1R15A (also known as growth arrest and DNA damage-inducible protein, GADD34), which is induced as part of the ISR, or by the constitutively expressed paralogue PPP1R15B (also known as constitutive repressor of eif2a phosphorylation, CReP) that is responsible for targeting the enzyme to eif2a (Fig 1) [15,76]. CReP normally operates in a complex with PP1c in unstressed cells to sustain translational homeostasis by maintaining low levels of eif2a phosphorylation [76]. In contrast, GADD34 expression is induced downstream of phosphorylated eif2a and ATF4 during the later stages of the ISR to significantly increase eif2a dephosphorylation [77]. Thus, the GADD34 PP1 complex acts as an important negative feedback loop to restore protein synthesis once the ER stress has been resolved, and as such aids in cell survival [78,79]. However, eif2a dephosphorylation may also be important to accommodate the translation of accumulated mrnas of stress-responsive genes during the ER stress and oxidative stress [16]. It may also facilitate the execution of cell death when cellular homeostasis cannot be restored, by allowing the synthesis of death-inducing proteins and also other proteins that accumulate in the cell and aggravate the proteotoxicity and oxidative stress [16,80]. The importance of eif2a dephosphorylation is highlighted by the fact that Ppp1r15a and Ppp1r15b-double-knockout mice exhibit early embryonic lethality, which can be rescued by eif2a S51A mutation that prevents eif2a phosphorylation [81]. Recently, in addition to PP1c and GADD34, G-actin was identified as a crucial and conserved component of the eif2a phosphatase complex, providing an insight into the importance of cytoskeletal dynamics in regulating the ER stress-induced ISR [82,83]. It is possible that the eif2a phosphatase complex contains other important regulatory factors that may differ among cell types or species. The mechanism of ISR termination may also involve CReP degradation as indicated by a recent study [84]. Interestingly, the role of GADD34 in a cell may not be limited to aiding cellular recovery after protein synthesis shut off, but it may also play an important, independent role in apoptosis induction [77,85,86]. Components of the ISR pathway eif2a at the core of the ISR eif2a, eif2b, and eif2c together form the eif2 complex and eif2a is the main regulatory subunit of this complex since it contains both the phosphorylation and RNA binding sites. Under normal conditions, eif2 plays a key role in the initiation of mrna translation and recognition of the AUG start codon [87,88]. It forms a ternary complex with GTP and Met-tRNA i that binds the 40S ribosome subunit, and together with two small initiation factors, eif1 and eif1a, it forms the 43S pre-initiation complex (PIC) [89,90]. The 43S PIC is recruited to the 5 0 methylguanine Cap of mrna in a process that is facilitated by the eif4f complex among others. The eif4f complex consists of the Cap-binding protein eif4e, eif4g that constitutes as a scaffold protein, and the RNA helicase eif4a [91]. The interaction between eif4g and eif3 further stabilizes the PIC, leading to its migration to the AUG start codon [89,90,92]. Upon binding of the Met-tRNA i anticodon and the AUG start codon, eif1 dissociates from the complex and GTP on eif2 is hydrolyzed with the aid of eif5. This leads to the dissociation of the eif2 GDP complex from the 40S ribosomal complex and its recycling for another round of initiation of mrna translation. Exchange of GDP for GTP is catalyzed by a guanine nucleotide exchange factor eif2b, and this converts eif2 back to its active form [87,88]. In response to ISR activation, phosphorylated eif2a blocks the eif2b-mediated exchange of GDP for GTP, thereby preventing the formation of the 43S PIC. This results in the global attenuation of 5 0 Cap-dependent protein synthesis and concomitant translation of selected mrnas that contain a short upstream open reading frame (uorf) in their 5 0 untranslated region (5 0 UTR). Examples of these preferentially translated mrnas include ATF4, ATF5, CHOP, and GADD34 (Fig 2) [92 94]. These mrnas do not require Cap recognition by the eif4f complex, and their translation relies on a re-initiation mechanism or the direct recruitment of ribosomes to internal ribosome entry sites (IRES) [6,95]. Phosphorylation of eif2a occurs at S51. Homozygous mutation at this site effectively prevents eif2a phosphorylation permitting normal mrna translation even under ER stress or glucose deprivation [96]. Early work showed that mice with homozygous mutation of the eif2a phosphorylation site die shortly after birth due to prolonged hypoglycemia, demonstrating the importance of translation initiation in glucose metabolism [96,97]. These data suggest that eif2a phosphorylation is crucial for sustaining proper physiological function of the liver and pancreas after birth, as eif2a phosphorylation is required for the induction of gluconeogenic enzymes and insulin [96]. Also, it is noteworthy that eif2a S51A/S51A MEF cells are hypersensitive to ER stress and require supplementation by nonessential amino acids and a reductive agent, which indicates that eif2a phosphorylation protects against metabolic and oxidative stress [96]. Moreover, cells with homozygous eif2a S51A mutation are unable to induce autophagy upon viral infection, starvation, and ER stress [98,99]. Thus, the phosphorylation of eif2a at S51 appears to be crucial for stress-induced autophagy (this will be discussed in a later section). ATF4 is the best characterized effector of the ISR ATF4 is a basic leucine zipper (bzip) transcription factor that belongs to the activating transcription factor/cyclic AMP response element binding protein (ATF/CREB family) [ ]. ATF4 has several dimerization partners that influence its regulation of gene transcription and can guide cellular outcome. In fact, ATF4 is a key deciding factor in cellular fate in response to ISR activation. It is regulated at the transcriptional, translational, and post-translational level, and moreover, its ability to interact with other transcription factors provides a further level of regulation. A consequence of this ª 2016 The Authors EMBO reports Vol 17 No

5 EMBO reports The integrated stress response Karolina Pakos-Zebrucka et al elf2α kinases 5'cap-dependent translation elf2 GTP P elf2 elf2b GDP ATF4 uorf1 uorf2 ATF4 CDS elf2b Met elf2 GDP Ternary complex Selective translation at coding DNA sequence ATF4 uorf1 uorf2 ATF4 CDS elf2α phosphatases Figure 2. Model for eif2-mediated translational control of ATF4. During the ISR, phosphorylation of eif2a subunit of eif2 complex inhibits eif2b-mediated exchange of eif2-gdp to eif2-gtp, thus reducing the formation of ternary complex consisting of eif2-gtp-methionyl-initiator trna. Under normal conditions, due to abundance of the ternary complex, ribosomes initiate scanning at upstream open reading frame uorf1 of ATF4 transcript and quickly re-initiate at uorf2. uorf2 overlaps out-of-frame with ATF4 coding DNA sequence (CDS) preventing ATF4 translation. During cell stress, limiting ternary complex availability leads to longer ribosomal scanning along the ATF4 transcript enabling re-initiation of translation at the ATF4 CDS. intricate regulation is that despite the common mediator, the ISR produces distinct tailored responses to different cellular stresses, with the activation of different ATF4 target genes also being highly dependent on stress intensity and the cellular context [2,101,103]. ATF4 functions as a transcription factor ATF4 has an important role in regulating both normal metabolic and redox processes as well as acting as a master transcription factor during the ISR. ATF4 has a vital role in many tissues and plays a central function in the regulation of obesity, glucose homeostasis, energy expenditure, and neural plasticity. Atf4-knockout mice reveal critical roles for ATF4 in embryonic lens formation [104,105], fetal liver hematopoiesis [106], and bone development [107]. Atf4-knockout mice are also smaller and leaner [106], suggesting a role for ATF4 in lipid metabolism. They have decreased fat mass caused by a reduction in the volume rather than the number of cells, suggesting that the deletion of ATF4 provides an increased fat mobilization and suppressed the fatty acid synthesis [108]. Knockout of Atf4 in mice has also revealed a physiological role in thermoregulation as these mice exhibit higher core body temperatures in response to cold stress [109]. ATF4 has also been implicated as a vital mediator of muscle weakness and atrophy in aging muscles as the reduction in its expression improves muscle quality, strength, and mass in mice [110]. Although the role of ATF4 in models of learning and memory formation is somewhat controversial, with both positive and negative effects suggested [ ], targeted knockdown of Atf4 in the mouse hippocampus indicated that it is necessary for memory formation and neural plasticity [115]. During stressful conditions, elevated translation of ATF4 mrna facilitates transcriptional upregulation of stress-responsive genes. ATF4 regulates the transcription of its target genes through binding to C/EBP-ATF response element (CARE) sequences that can mediate the transcriptional activation in response to various stimuli [ ]. During amino acid starvation, these sequences function as amino acid response element (AARE) and ATF4 is the only transcription factor known to bind all known AARE sequences [103,119,120]. ATF4 can form homodimers and heterodimers with several other bzip transcription factors, including its downstream target CHOP [100,120]. Recent studies have connected ATF4 and CHOP with autophagy induction in mammalian cells [6,103,121]. ATF4 and/or CHOP can bind to the specific promoter cis element in their target genes, leading to the induction of the AARE-dependent transcription of genes involved in autophagy activation upon starvation [103]. Moreover, the formation of an ATF4 CHOP heterodimer increases its binding affinity for AARE and this is reported as an early event in transcriptional induction of autophagy genes [103]. Upon ER stress and amino acid depletion in mouse cells, ATF4 alone, or together with CHOP, preferentially binds to proximal promoter regions of target genes, including Atf3, Ppp1r15a, Trib3, Wars, and Rpl7 [122]. Although it is well established that ATF4 alone regulates the expression of genes involved in amino acid transport and biosynthesis, Kaufman and colleagues showed that in response to ER stress, ATF4 and CHOP interact to regulate common genes involved in cellular amino acid metabolic processes, mrna translation, and the unfolded protein response (UPR) [122]. Although ATF4 mainly acts as a transcriptional activator of the cohort of genes involved in cellular stress adaptation, it can also act as a transcriptional repressor [100]. Some examples of the repressor activity of ATF4 include repression of CRE-dependent transcription of genes such as enkephalin and chromogranin B [101,123]. The repressive effect of ATF4 on transcription can be linked to the inhibition of induction and maintenance of long-term memory by ATF4 [124]. ATF4 is implicated in amino acid metabolism through the induction of asparagine synthetase (ASNS) during both the amino acid and UPR signaling where it binds promoters that contain nutrient-sensing response elements 1 and 2 (NSRE-1 and NSRE-2) [120]. Together, these two elements function as an enhancer and mediate the activation of the ASNS gene in response to ATF4, regardless of whether the initial stimulus was amino acid limitation or ER stress [120,125]. ATF4 was identified as a major regulator of VEGF expression induced by oxidized phospholipids, a common lipid component of atheroma, thus implicating ATF4 as a pathogenic factor in vascular damage [126]. Recently, several studies have shown ATF4 to be a link between stresses such as glucose starvation or ER stress and mammalian target of rapamycin (mtor) inhibition through upregulation of cytoprotective Sestrin2 [ ]. ATF4 acts in combination with other proteins Due to the presence of a leucine zipper domain, ATF4 can interact with other proteins 1378 EMBO reports Vol 17 No ª 2016 The Authors

6 Karolina Pakos-Zebrucka et al The integrated stress response EMBO reports forming homodimers and/or heterodimers. When ATF4 is not bound to its DNA target, it exists as a monomer [130]. Transcriptional selectivity of ATF4 is modulated by the formation of heterodimers with other bzip or AP-1 members. These interactions of ATF4, with other transcription factors or interacting partners, influence the outcome of ISR signaling; for example, interactions with ATF3 enhance cellular efforts to re-establish homeostasis, while interactions with CHOP promote cell death upon ER stress [131,132]. Consequently, by association with other proteins and transcription factors, ATF4 can modulate the transcriptome of the cell in response to diverse stress stimuli. Some interactors, such as PHD1 [133], PHD3 [134] during hypoxia, and TRIB3 [135] upon ER stress and amino acid starvation, can effectively decrease ATF4 transcriptional activity. Interestingly, TRIB3 is a downstream target of ATF4, and thus, its activation provides a negative feedback loop to control ATF4 activity during ER stress and amino acid deprivation [131,135]. The interacting partners that cooperate with ATF4 are summarized in Table 1. It seems that the interaction between ATF4 and a precise set of bzip family members and co-activators regulates specific gene transcription, deciding on the signaling outcome. This interaction network must be therefore highly coordinated and time dependent to induce and modulate gene transcription tailored to the cellular demand. Translational regulation of ATF4 The primary mechanism by which the levels of ATF4 protein are adjusted in response to different stresses, such as ER stress, hypoxia, or oxidative stress, is through translational regulation [17,136,137]. The mechanism is highly conserved, with yeast regulating levels of Gcn4p (a functional homologue of ATF4) using a very similar mechanism [138,139]. Since yeast do not have PERK, global inhibition of translation is related to the activation of Gcn2p (the yeast homologue of GCN2) and eif2a phosphorylation upon amino acid depletion [139]. This results in preferential synthesis of Gcn4p, a transcription factor that controls the expression of genes involved in amino acid synthesis and transport [138]. Translational control of yeast GCN4 requires four uorfs [140]. When the ternary complex is abundant, ribosomes initiate scanning at uorf1 and re-initiate at inhibitory downstream uorfs that precludes the translation of GCN4 coding sequence (CDS). Under amino acid depletion, more time is required for the ribosome to re-initiate translation, due to low levels of ternary complex, so that ribosomes scan through uorf4 and re-initiate at the GCN4 CDS [141]. In mammals, mouse Atf4 has features similar to the GCN4 [13]. The mouse gene contains two uorfs (uorf1 and uorf2) that are located 5 0 to the Atf4 CDS (Fig 2), while human ATF4 mrna contains three upstream open reading frames (uorf1, uorf2, and uorf3) [142]. Under normal cellular conditions, the translation of mouse Atf4 mrna is initiated at uorf1, which encodes a peptide just three amino acids in length, and re-initiates at uorf2. This precludes translation of Atf4 mrna because, unlike GCN4, the uorf2 (uorf3 in human) sequence overlaps with the Atf4 CDS in an out-of-frame manner [100, ]. In stressed cells, ribosomes scan through uorf2 and re-initiate at the Atf4 CDS [5,143]. Thus, upon eif2a phosphorylation, re-initiation of translation at the Atf4 CDS results in approximately fivefold higher ATF4 expression (Fig 2) [13,87,143]. However, Walter s group recently observed high levels of peptide synthesis from both ATF4 uorfs during ER stress and oxidative stress conditions [145]. These authors suggest that this deviation from the current re-initiation model may indicate that either only a few ribosomes skip uorf2 or that a combination of re-initiation and scanning past uorf2 ( leaky scanning ) occurs. Interestingly, apart from the ribosome re-initiation mechanism, in HeLa and THP-1 cells the translation of an alternatively spliced variant of ATF4 mrna is reported to be also controlled by an IRES sequence in 5 0 UTR [95]. Transcriptional regulation of ATF4 Under non-stressed conditions, the levels of ATF4 mrna transcripts available are low [87]. ATF4 gene transcription is induced in response to different stresses and is mediated by different transcription factors [17]. For example, nuclear factor-like 2 (NRF2) induces ATF4 in response to oxidative stress [126], CLOCK regulates ATF4 expression in response to the chemotherapeutic drugs cisplatin and etoposide [146], TFEB and TFE3 induce ATF4 transcription under ER stress and starvation conditions [147], and a pancreas-specific transcription factor (PDX1) mediates transcriptional induction of Atf4 during ER stress in mouse islet b cells [148]. Interestingly, in a positive feedback loop, ATF4 downstream gene targets, such as NUPR1, can also elevate ATF4 mrna levels [149]. In contrast, hypoxia does not alter ATF4 mrna levels, although it can increase ATF4 levels by translational and post-translational mechanisms [136]. Furthermore, there is evidence of transcriptional repression of ATF4 by some cellular stressors. For example, C/EBPb, a member of the C/EBP family, has been identified as a transcriptional repressor of Atf4 during UV irradiation, revealing a novel role for C/EBPb in regulating the ISR [150]. Thus, despite an increase in eif2a phosphorylation upon UV irradiation, ATF4 induction is not observed because of diminished levels of available ATF4 transcript [150,151]. Transcriptional repression of ATF4 is also observed in nonalcoholic fatty liver and nonalcoholic steatohepatitis [152]. ATF4 post-translational modifications and protein stability ATF4 is an unstable protein with a half-life of < 1 h under normal conditions [102,153,154]. Several post-translational modifications regulate ATF4 stability, including phosphorylation and ubiquitination, which are the two most significant in this regard (Table 2). ATF4 degradation is mediated by the ubiquitin-proteasome pathway [153]. Proteasomal inhibitors such as bortezomib and MG132 cause an increase in ATF4 protein levels as a dual consequence of inhibited proteasomal degradation (which stabilizes the protein), and ISR activation (which is triggered by the depletion of intracellular amino acids as a result of proteasome inhibition) [153, ]. Phosphorylation of ATF4 at S219 (within its degradation motif sequence DSGICMS) is essential for its interaction with the F-box protein b-transducin repeat-containing protein (b-trcp), the receptor component of SCF E3 ubiquitin ligase complex (SCF btrcp ) that targets ATF4 for proteasomal degradation (Fig 3) [153]. Also, phosphorylation of ATF4 at S224 within the same motif, and proline-directed phosphorylation at multiple proline residues surrounding b-trcp have been implicated in regulating ATF4 interaction with b-trcp, indicating the tight control of ATF4 degradation [153,158]. Phosphorylation of ATF4 at S245 by ribosomal protein S6 kinase a-2 (RSK2) or S254 by protein kinase A (PKA) can enhance its transcriptional activity during osteoblast and osteoclast differentiation [107,159]. In addition, there are several other kinases that have been shown to interact with ATF4, for example, zipper-interacting ª 2016 The Authors EMBO reports Vol 17 No

7 EMBO reports The integrated stress response Karolina Pakos-Zebrucka et al Table 1. ATF4 interacting partners. Interacting partner Name Function References b-trcp F-box protein b-transducin repeat-containing Interaction of b-trcp with ATF4 controls ATF4 stability and [153] protein inhibits its transcriptional activity CENP-F Centromere protein F Interacts with ATF4 and negatively regulates ATF4 [256] transcriptional activation in interphase CEP290 Centrosomal protein of 290 kda Interacts with ATF4 and induces its transcriptional activity [257] CHOP C/EBP homologous protein Interaction between ATF4 and CHOP results in negative regulation of ATF4-dependent transcriptional activation of the ASNS gene ATF4 and CHOP interact to induce target genes involved in protein synthesis and the UPR [122,258] PHD3 Prolyl hydroxylase 3 Regulates ATF4 protein stability [134] FAM175B (Abro1) BRISC complex subunit Abro1 Interaction with ATF4 is crucial for the cytoprotective function of Abro1 in response to oxidative stress FIAT Factor inhibiting ATF4-mediated transcription Modifies early osteoblast activity by interacting with ATF4 [260] FOS Proto-oncogene c-fos Forms heterodimers with ATF4 [261] Fra1 Fos-related antigen 1 Forms heterodimers with ATF4 [261] GABA(B) Gamma-aminobutyric acid type B Interacts with ATF4 in mouse brain during the postnatal development GPE-1 GPE1-binding protein Forms heterodimers with ATF4 that regulate G-CSF gene expression HIF1A Hypoxia inducible factor 1-alpha Interaction with ATF4 initiates bone angiogenesis in hypoxic osteoblasts Jun Proto-oncogene c-jun Forms heterodimers with ATF4 [261] NIPK Neuronal cell death-inducible putative kinase (TRIB3) Interacts with ATF4 and inhibits ATF4 transcriptional activity Nrf-2 Nuclear factor erythroid 2-related factor 2 Interaction between ATF4 and Nrf2 regulates the expression of heme oxygenase-1 gene p300 Histone acetyltransferase p300 Interaction with p300 stabilizes ATF4 and increases its transcriptional activity RPB3 RNA polymerase II subunit 3 Interaction with RPB3 enhances ATF4 transcriptional potential in muscle differentiation RSK2 (RPS6KA3) Ribosomal protein S6 kinase alpha-2 ATF4 and RSK2 interact to regulate osteoblast differentiation and function RUNX2 Runt-related transcription factor 2 Interaction with ATF4 stimulates osteoblast differentiation and bone formation SATB2 DNA-binding protein SATB2 Regulates skeletal development and osteoblast differentiation through the direct interaction with ATF4 and enhances its activity SKIP3 Orthologue of the Drosophila TRIB3 and rat NIPK Interaction between SKIP3 and ATF4 is associated with ATF4 proteolysis Tax of HTLV-1 Trans-activating transcriptional regulatory protein of HTLV-1 ATF4 interacts with Tax and triggers Tax transactivation of HTLV TRIB3 Tribbles homolog 3 Interacts with ATF4 and negatively regulates the ATF4-dependent transcription. Zhangfei HCF-binding transcription factor Zhangfei Forms heterodimers with ATF4 and enhances its transcriptional activity ZIP Zipper-interacting protein kinase Forms heterodimers with ATF4, which inhibits the apoptotic activities mediated by ZIP [259] [262] [263] [264] [265] [266] [164] [267] [107] [268] [269] [161] [270] [135,197] [271] [160] protein kinase (ZIPK) [160], casein kinases 1 and 2 (CK1 and CK2) [158], and SKIP3 [161], but their full role in regulating ATF4 activity in response to physiological stimuli and hypoxia, respectively, remains to be addressed. In addition to phosphorylation during ER stress, ATF4 stability and activity can be negatively regulated by prolyl hydroxylase domain protein 3 (PHD3) leading to hydroxylation at proline residues 60 and 235 [162]. This process is regulated by Siah 1/ EMBO reports Vol 17 No ª 2016 The Authors

8 Karolina Pakos-Zebrucka et al The integrated stress response EMBO reports Table 2. ATF4 post-translational modifications and their role. Modification Residue Function References Acetylation K311 Inhibition of ATF4 [164] ubiquitination Hydroxylation P60 P235 Possible decrease in ATF4 transcriptional activity [162] Phosphorylation Ubiquitination S219 S224 S245 S251 S254 ubiquitin ligases that target PHD1 and PHD3 for degradation and thus increase ATF4 stability [162]. These findings demonstrate an important link between phosphorylation and hydroxylation, as P235 is localized close to the b-trcp recognition DSGICMS motif [162]. Interaction of ATF4 with PHD1, but not with PHD3, is a stabilizing mechanism for ATF4. However, both interactions lead to a decrease in ATF4 transcriptional activity [133,134,163]. Additionally, ATF4 stability can be enhanced by histone acetyltransferase p300, independently of its catalytic activity, resulting in the inhibition of ATF4 ubiquitination [164]. Cellular outcome of the ISR btrcp-dependent degradation Upregulation of ATF4 transcriptional activity [153,272] [107,159] S215 ATF4 stabilization [273] S219 D218 ATF4 degradation [153] Although multiple stresses converge on eif2a phosphorylation to activate the ISR, the cellular outcome is not always the same. The effect of ISR activation depends not only on the nature of the stress, its duration and severity, but also on the extent of eif2a phosphorylation and translation of ATF4 mrna and other bzip transcription factors [17,165]. It is commonly accepted that a short-lived ISR is an adaptive, pro-survival response aiming at resolving stress and restoring homeostasis, while a prolonged ISR can signal toward cell death induction [154]. Therefore, this dual effect of eif2a p300 interaction site N-terminal domain Oxygen dependent degradation domain βtrcp recognition motif DSGICMS Basic domain ATF4 Leucine zipper C-terminal domain Figure 3. Schematic ATF4 protein structure. ATF4 protein consists of 351 amino acids organized into several domains and motifs. It contains basic domain for DNA binding and leucine zipper domain involved in protein protein interactions. ATF4 has an oxygen-dependent degradation domain, which is recognized by PHD3. ATF4 has b-trcp recognition motif, which is a degradation motif. Depending on the phosphorylation events mainly at S219, ATF4 is recognized by b-trcp and targeted for proteasomal degradation. phosphorylation raises an important question concerning how the switch between pro-survival and pro-death signaling by ISR is regulated, as well as whether a threshold of cell stress signals exists that favors the activation of cell death proteins. Cellular recovery signaling by the ISR The ISR, together with other cellular adaptation pathways, functions as an important part of the cellular defense strategy in response to stress. It does this mainly through altering global protein synthesis and through the regulation of genes that promote pro-survival signaling such as through the activation of autophagy, or that counteract pathways that lead to cell death such as apoptosis or proteotoxicity (impairment of cell function due to the effects of misfolded proteins). Notably, there is also a cross-talk between the ISR and other pro-survival pathways such as the UPR, phosphatidylinositol-3 kinase (PI3K) signaling, autophagy, and the ubiquitin-proteasome system. These are discussed below. One of the major effects of the ISR is on protein synthesis. The initial repression of global mrna translation plays a very important role in promoting cell survival in the face of different stresses that activate the ISR. The accumulation of unfolded proteins in the ER induces a condition of ER stress, which is relieved by the reduced level of incoming proteins when global protein synthesis is inhibited [1]. PKR activation of the ISR during viral infection helps to reduce the translation of viral mrnas, thus protecting the cells [8,42]. Under conditions of amino acid depletion, activation of the ISR by GCN2 reduces the need for amino acids for protein synthesis, thus alleviating this stress [34]. Activation of the ISR by HRI under conditions of low heme lessens the need for heme by attenuating the translation of globin mrnas, thus reducing the stress and promoting survival [52]. It is important, however, to note that timely termination of the ISR also plays a key role in promoting long-term cell survival, by re-starting synthesis of essential proteins. This is achieved through dephosphorylation of eif2a by the phosphatase GADD34, which is induced by ATF4 and its downstream targets CHOP and ATF3 [15,78,79,166]. Through the activation of macroautophagy, commonly known as autophagy, the ISR can regulate cell survival and cell death pathways. Autophagy promotes the bulk removal and degradation of unfolded proteins or aggregates as well as damaged organelles while also serving as a means to replenish depleted amino acids for building proteins and to provide energy to a starved cell. Although the precise mechanisms by which phosphorylated eif2a leads to autophagy are still poorly understood, it is interesting to note that distinct intrinsic and extrinsic stresses that lead to the phosphorylation of eif2a have also been shown to induce autophagy. For example, ER stress leads to an increased phosphorylation of eif2a and the subsequent upregulation of certain autophagy receptors such as SQSTM1, NBR1, and BNIP3L in a PERK-dependent manner [167]. Pharmacological inhibition of PERK suppressed transcriptional upregulation of these autophagy receptors in mammalian cells [167]. In addition, PERK-mediated phosphorylation of eif2a mediates increased ATG12 and LC3 conversion upon the expression of polyq72 aggregates in C2C5 cells [98]. In fact, the PERK arm of the UPR can regulate all stages of autophagy including induction, vesicle nucleation, phagophore elongation, and maturation [168]. Consistent with this, another study showed that ER stress caused by bluetongue virus induces autophagy through a PERK-eIF2a pathway ª 2016 The Authors EMBO reports Vol 17 No

9 EMBO reports The integrated stress response Karolina Pakos-Zebrucka et al in infected cells [169]. In that study, both pharmacological inhibition of PERK activity and genetic silencing of eif2a were shown to significantly reduce the level of the autophagy marker LC3 [169]. Furthermore, the induction of autophagy during hypoxia is also dependent on PERK-mediated eif2a phosphorylation, as the disruption of PERK signaling or exposure of non-phosphorylatable eif2a S51A mutant to hypoxia decreases the levels of MAP1LC3B and ATG5 transcripts [121]. It has also been shown that GCN2-mediated phosphorylation of eif2a is essential for the induction of autophagy upon amino acid starvation in tumor cells [7]. While GCN2- knockout cells displayed the reduced level of LC3, the eif2a S51Amutant cells could not induce LC3 processing [7]. Similarly, S51 of eif2a has also been demonstrated to be important for amino acid starvation-induced autophagy in yeast and MEFs [99]. Together, these findings place eif2a phosphorylation as the central link between different stresses and induction of autophagy. Downstream of eif2a phosphorylation, ATF4 has been shown to be required for autophagy induction. However, it has been suggested that mechanisms directed from eif2a phosphorylation other than selective translation of ATF4 mrna might also be involved in the activation of autophagy [170]. During hypoxia, ER stress, or amino acid deprivation, there is ATF4-dependent transcriptional upregulation of essential autophagy genes involved in autophagosome biogenesis and function, for example, MAP1LC3B and ATG5 [6,103,167]. ATF4 can also lead to an increased expression of regulated in development and DNA damage response 1 (REDD1; also known as Ddit4), which suppresses mtor complex 1 (mtorc1) activity leading to autophagy induction upon ER stress and starvation [171,172]. ATF4 activation due to amino acid deprivation stimulates the upregulation of several genes involved in autophagy including Atg3, Atg5, Atg7, Atg10, Atg12, Atg16, Becn1, Gabarap, Gabarapl2, Map1lc3b, and Sqstm1 [103]. Through the upregulation of key autophagy genes, the ISR contributes to increased autophagic flux that helps the cell to deal with the stress caused by hypoxia and nutrient deprivation through the enhanced recycling of cytoplasmic components and maintenance of cellular biosynthetic capacity and ATP levels, for example, by supplying amino acids for de novo protein synthesis and providing substrates for the tricarboxylic acid cycle, such as amino acids and free fatty acids [7,173]. It is worth noting that different autophagy genes can have different levels of dependence on ATF4 and CHOP signaling and that the activation of such genes is determined by the ratio of ATF4 and CHOP proteins that are bound to specific promoter cis elements, thus allowing subtle tuning of gene expression that can be tailored specifically to meet cellular needs [103]. Interestingly, a cytoprotective function of eif2a phosphorylation against conditions that mimic viral infection or induce ER stress has been reported to occur through the activation of pathways that promote cell survival, such as PI3K and its downstream target Akt/mTOR [174]. In this regard, it is also of interest to consider the effect of proteasome inhibition on survival signaling by the ISR. Proteasome inhibition, for example, using bortezomib, leads to the activation of the ISR through GCN2 as a result of depletion of amino acids for protein synthesis [157]. In mammalian cells, the depletion of amino acids due to proteasome inhibition also activates autophagy through mtor in an attempt to restore amino acid levels [157]. Supplementation with critical amino acids that are depleted by proteasome inhibition attenuates eif2a phosphorylation and suppresses autophagy [157]. Thus, amino acid depletion constitutes a link between autophagy induction and ISR activation in an attempt to promote the survival of cells. The UPR that is activated by ER stress is mainly an adaptive response and encompasses the activation of the ISR. In this pathway, PERK, which phosphorylates eif2a to activate the ISR, functions in concert with the other arms of the UPR, IRE1, and ATF6, to ameliorate proteotoxicity by enhancing the transcription of genes that promote protein folding and degradation of misfolded or aggregated polypeptides [175,176]. The interplay between the different arms of the UPR controls the cellular outcome [177]. The survival benefit provided by the ISR during ER stress reveals ATF4 as a focal point linking PERK-mediated translational control with IRE1- and ATF6-mediated gene expression [1]. It has been proposed that the relative duration of PERK and IRE1 signaling dictates the cellular outcome, with the sustained PERK activation promoting apoptosis and the prolonged IRE1 activation leading to cell survival [178,179]. The ISR also promotes survival signaling by negative regulation of cell death pathways, particularly apoptosis. For example, in response to ER stress, PERK-mediated activation of the ISR leads to an upregulation of cellular inhibitor of apoptosis proteins (ciap1 and ciap2) in both tumor-derived cell lines and non-tumor cells [ ]. ATF4 has also been shown to mediate bortezomibinduced upregulation of anti-apoptotic MCL-1 leading to resistance to the drug [183]. Both MCL-1 and ciaps can inhibit apoptosis at different points in the pathway that are upstream and downstream of the release of cytochrome c from the mitochondria (a critical step in the intrinsic apoptosis pathway). PERK can also mediate a prosurvival role by the induction of microrna 211 (mir-211), which attenuates a stress-dependent expression of CHOP and which was suggested by the authors as a mechanism that allows cells to reestablish homeostasis before the initiation of apoptosis [184]. However, despite having a beneficial role in restoring homeostasis, these mechanisms could also contribute to tumor development. Increased expression of mir-211 was detected in two mouse models of mammary carcinoma as well as in human B-cell lymphoma and was found to be PERK dependent [184]. Several other genes activated during ISR appear to contribute to pro-survival signaling, although their importance and function in determining cellular outcome remains to be fully addressed. An example is IBTKa, the a isoform of inhibitor of Bruton s tyrosine kinase, an important substrate adaptor for protein ubiquitination, which has emerged recently as an important pro-survival factor activated during ER stress [185]. Translational repression by eif2a has also been implicated in nuclear factor kappa light chain enhancer of activated B cells (NF-jB) pathway induction to protect against ER stress [186]. ATF4 also activates the transcription factor NUPR1, which regulates the expression of metabolic stress-responsive genes, especially those involved in DNA repair and cell cycle regulation, and therefore may be considered as a pro-survival factor [149,187]. Cell death signaling by the ISR Within the ISR are embedded pathways that can lead to the induction of cell death if the adaptive response does not restore homeostasis. These are mainly regulated through the transcriptional activity of ATF4 and some of its downstream targets, particularly CHOP and ATF3. Post-transcriptional regulation of gene expression through the upregulation of micrornas also plays a role in cell death signaling by the ISR. These will be discussed below EMBO reports Vol 17 No ª 2016 The Authors